Heterogeneity of cells in tumors appears to be a major obstacle in the successful treatment of cancer. It is also of importance in understanding the progression of cells to autonomous growth including metastasis. The highest estimates of the rate of variation responsible for heterogeneity have been on the order of one heritable alteration per 105 cell divisions. In the past yearwe were able to isolate five clones among the progeny of a Balb/3T3 cell which had just undergone spontaneous transformation and to characterize their properties. We found that each of the clones could be distinguished from the others by morphology, colony-forming efficiency in agar (CFEag), and rate of tumor formation in nude mice. The implication of this finding is that cultured cells are so unstable soon after transformation that essentially every cell may differ from the others in one or more characteristics, i.e., that there is a truly radical heterogeneity. We have also studied the evolution of further changes in these properties among the five clonal populations over a 6-month period and conclude that the type of variation which led to the initial differences among the clones could also be responsible for continuing change akin to tumor progression. Since the frequency of this type of variation is very high and involves several properties in a quantitative but uncoordinated way, it is unlikely to be caused by conventional genetic mutations. Rather, it is likely to be an epigenetic event. This type of variation is also likely to play a role in our second major finding, which is that some properties of transformed cells are altered during tumor formation in the nude mouse and again when the tumor cells are cultured. The former contradicts the generally accepted view that cells are not changed by passage through nude mice. We find our example, that a decrease in CFEag of more than 100-fold, is frequently observed during tumor formation. Cells from some tumors regain their original high CFEag after repeated passaging in culture but others do not. That is, there is a heterogeneity of response, some changes being stable and others unstable. The evidence to date indicates the alterations in cells during tumor formation and subsequent cultivation are basically epigenetic in nature and similar in type to those described above which occur after transformation. The results promise to provide new ground for understanding the origin and progression of cancer. (J)